DNA denaturation sensitivity may invalidate bromodeoxyuridine-DNA flow cytometric analysis of potential doubling times in colorectal tumours

Background Flow cytometric analysis of 5-bromo-2'-deoxyuridine (BrdU)-substituted DNA has been used to calculate tumour cell proliferation rate. In this methodology, DNA denaturation, commonly by hydrochloric acid, is essential to expose incorporated BrdU for quantification with monoclonal antibodies. This study was designed to establish the validity of this technique by examining the change in flow cytometric DNA profiles introduced by DNA denaturation procedures. Methods Four experiments were performed using suspensions of nuclei derived from human colorectal tumours exposed in vivo to 150 mg/m 2 BrdU 8-11 h before sampling. Results After denaturation with hydrochloric acid 2 mol/l a significant decrease was observed in the DNA aneuploid G1 population (P<0.00l) with a concurrent increase in the DNA aneuploid S phase fraction (P<0.05). These changes were independent of the washing-centrifugation step and were maximal at different hydrochloric acid concentrations for different tumours. Conclusion Hydrochloric acid denaturation introduces a tumour-specific non-linear variation in the analysis of BrdU.